The present invention relates to methods useful in the administration of gene products to animals using adenoviral vectors.
Modified viruses have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral vector systems present several advantages for such uses. Adenoviruses are generally associated with benign pathologies in humans, and the 36 kb of the adenoviral genome has been extensively studied. Adenoviral vectors can be produced in high titers (e.g., about 1013 pfu), and such vectors can transfer genetic material to nonreplicating, as well as replicating, cells (in contrast with, for example, retroviral vectors which only transfer genetic material to replicating cells). The adenoviral genome can be manipulated to carry a large amount of exogenous DNA (up to about 8 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene Ther., 3, 147-154 (1992)). Additionally, adenoviruses generally do not integrate into the host cell chromosome, but rather are maintained as a linear episome, thus minimizing the likelihood that a recombinant adenovirus will interfere with normal cell function. Aside from being a superior vehicle for transferring genetic material to a wide variety of cell types, adenoviral vectors represent a safe choice for gene transfer, a particular concern for therapeutic applications.
A variety of recombinant adenoviral vectors have been described. Most of the vectors in use today derive from the adenovirus serotype 5 (Ad5), a member of subgroup C. An exogenous gene of interest typically is inserted into the early region 1 (E1) of the adenovirus. Disruption of the E1 region decreases the amount of viral proteins produced by both the early regions (DNA binding protein) and late regions (penton, hexon, and fiber proteins), preventing viral propagation. These replication deficient adenoviral vectors require growth in either a complementary cell line or in the presence of an intact helper virus, which provides, in trans, the essential E1 functions (Berker et al., J. Virol., 61, 1213-1220 (1987); Davidson et al., J. Virol., 61, 1226-1239 (1987); Mansour et al., Mol. Cell Biol., 6, 2684-2694 (1986)). More recently, adenoviral vectors deficient in both E1 and the early region 4 (E4) have been used to substantially abolish expression of viral proteins. In order to insert the larger genes (up to 8 kb) into the adenoviral genome, adenoviral vectors additionally deficient in the nonessential early region 3 (E3) are used. Multiply deficient adenoviral vectors are described in published PCT patent application WO 95/34671.
One limitation of adenoviral vector systems is the ability of the adenoviral vector to transduce a wide variety of proliferating and quiescent cells (Michou et al., Gene Ther., 4, 473-482 (1997)). This ability, while a benefit in transducing the target area, is a limitation when the adenoviral vector xe2x80x9cleaksxe2x80x9d out of the targeted area and transduces other cells it contacts. Tranduction of the surrounding cells is a severe problem when the gene product encoded by the adenoviral vector is harmful, toxic, or otherwise undesirable with respect to these non-targeted areas.
Another limitation of the adenoviral vector system is the cellular and humoral immune response generated within the host animal. Initial administration elicits a reaction from both CD8+ and CD4+ T cell lymphocytes which eliminate virus infected cells within 28 days after infection, limiting the duration of the transgene expression. In addition, neutralizing antibodies produced by B lymphocytes in cooperation with CD4+ cells inhibit the effectiveness of a repeat administration of the adenoviral vector. Proliferation and specificity of the antibodies is achieved through interactions between the adenoviral vector, B-cell surface immunoglobulins and activated CD4+ surface proteins (particularly CD40Li, which binds CD40 on the surface of the B cell) (Yang et al., J. Virol., 69, 2004 (1995)).
Attempts to circumvent the humoral immune response to allow repeat administration of the adenoviral vector have met with limited success. These attempts have been focused in two areas, immunosuppression and alteration of the adenoviral vector. Several groups have experimented with various immunosuppressant drugs or antibodies specific for CD4+, CD40 ligand, or CTLA4Ig to reduce the adenovirus-specific humoral immune response (Lee et al., Hum. Gene Ther., 7, 2273 (1996) (CD4+); Yang et al., J. Virol., 70, 6370 (1996) (CD40 ligand); Kay et al., Nature Gen., 11, 191 (1995) (CTLA4Ig)). Although some of these results have been encouraging, there is a substantial risk associated with systemic immune suppression in a clinical setting.
In another study, subretinal administration of an adenoviral vector containing the bacterial xcex2-galactosidase gene resulted in minimal circulating antibodies specific to the adenoviral vector. This was most likely a reflection of the immune privileged status of the retina. Although there was minimal retinal toxicity to the adenovirus, several of the animals injected developed localized granulomatous infiltrate at the injection site (Bennett et al., Hum. Gene Ther., 7, 1763-1769 (1996)). Subretinal administration is not an option for many applications where adenoviral vectors are employed.
Alteration of the adenoviral vector is time consuming and has not been entirely successful in sufficiently attenuating the immune response. Limited readministration of the adenoviral vector has been accomplished when adenoviral vectors of different serotypes within the same subgroup are used; however, persistence of expression of the transgene was not comparable to the initial administration (Mack et al., Hum. Gene Ther., 8, 99-109 (1997)).
Accordingly, there is a need for improved methods of administering adenoviral vectors to animals, particularly, to prevent leakage of the adenoviral vector from the target area and to circumvent the humoral immune response elicited by adenoviral vectors. The present invention provides such methods. This and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a method of targeting a gene product in a particular muscle of an animal. The method utilizes systemic neutralizing antibodies to neutralize an adenoviral gene transfer vector containing an exogenous gene outside the particular muscle. The adenoviral gene transfer vector is administered such that the exogenous gene is expressed and the gene product is produced only in the particular muscle of administration.
The present invention further provides a method of producing a gene product in a skeletal muscle of an animal. The method comprises a first intramuscular administration of an adenoviral vector to the skeletal muscle of an animal, and a second administration of an adenoviral gene transfer vector containing an exogenous gene encoding a gene product. Administration is such that the exogenous gene is expressed and the gene product is produced in the skeletal muscle of the animal.
The invention may best be understood with reference to the accompanying drawings and in the following detailed description.